Department of Chemistry, Columbia University, 550 West 120th Street, MC 4854, NWC Building, New York, New York 10027, USA.
Acc Chem Res. 2011 Sep 20;44(9):784-92. doi: 10.1021/ar200099f. Epub 2011 Aug 31.
To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, we now face the challenge of understanding how macromolecular machines, signaling pathways, and other biological networks operate in the complex environment of the living cell. The fluorescent proteins (FPs) revolutionized our ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biology, they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chemical methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chemical tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chemical tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an organic fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chemical tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, we focus on the development and application of chemical tags for studying protein function within living cells. Thus, in our overview of different chemical tagging strategies and technologies, we emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. We highlight recent applications in which the chemical tags have enabled sophisticated biophysical measurements that would be difficult or even impossible with FPs. Finally, we conclude by looking forward to (i) the development of high-photon-output chemical tags compatible with living cells to enable high-resolution imaging, (ii) the realization of the potential of the chemical tags to significantly reduce tag size, and (iii) the exploitation of the modular chemical tag label to go beyond fluorescent imaging.
为了在前一个世纪在理解试管中单个蛋白质的作用方面取得的巨大进展的基础上更进一步，我们现在面临着理解大分子机器、信号通路和其他生物网络如何在活细胞的复杂环境中运作的挑战。荧光蛋白 (FP) 通过对荧光标签进行基因编码，使单个蛋白质能够被选择性标记，从而彻底改变了我们在细胞中直接研究蛋白质功能的能力。尽管 FP 仍然是细胞生物学的宝贵工具，但面对现在越来越需要的对蛋白质相互作用进行日益复杂的动态测量以揭示细胞机制的情况，它们显示出了局限性。因此，正如上个世纪用于在试管中选择性标记蛋白质的化学方法对体外生物物理学产生了重大影响一样，化学标记技术现在正准备提供一个突破，以应对本世纪在活细胞中理解蛋白质功能的挑战。通过化学标记，感兴趣的蛋白质被连接到多肽上，而不是 FP 上。然后，多肽被用有机荧光团或其他探针修饰。FlAsH 肽标签于 1998 年首次报道。此后，更精细的蛋白质标签，如 TMP-和 SNAP-标签，提高了选择性，并能够以高信噪比对细胞内蛋白质进行成像。为了实现对强大荧光团的直接掺入，仍需要进一步改进，但酶介导的化学标签显示出克服选择性标记短肽标签的困难的希望。在本说明中，我们重点介绍了用于研究活细胞内蛋白质功能的化学标签的开发和应用。因此，在我们对不同的化学标记策略和技术的概述中，我们强调了使标记反应具有足够选择性和荧光探针具有足够良好行为以高信噪比对细胞内蛋白质进行成像的挑战。我们重点介绍了最近的应用，其中化学标签使复杂的生物物理测量成为可能，而这些测量使用 FP 则是困难甚至不可能的。最后，我们展望未来：(i) 开发与活细胞兼容的高光子输出化学标签，以实现高分辨率成像；(ii) 实现化学标签显著减小标签尺寸的潜力；(iii) 利用化学标签的模块化标签超越荧光成像。
